Yellina et al. EvoDevo 2010, 1:13
http://www.evodevojournal.com/content/1/1/13
RESEARCH
Open Access
Floral homeotic C function genes repress specific
B function genes in the carpel whorl of the basal
eudicot California poppy (Eschscholzia californica)
Aravinda L Yellina1, Svetlana Orashakova1, Sabrina Lange1, Robert Erdmann1, Jim Leebens-Mack2, Annette Becker1*
Abstract
Background: The floral homeotic C function gene AGAMOUS (AG) confers stamen and carpel identity and is
involved in the regulation of floral meristem termination in Arabidopsis. Arabidopsis ag mutants show complete
homeotic conversions of stamens into petals and carpels into sepals as well as indeterminacy of the floral
meristem. Gene function analysis in model core eudicots and the monocots rice and maize suggest a conserved
function for AG homologs in angiosperms. At the same time gene phylogenies reveal a complex history of gene
duplications and repeated subfunctionalization of paralogs.
Results: EScaAG1 and EScaAG2, duplicate AG homologs in the basal eudicot Eschscholzia californica show a high
degree of similarity in sequence and expression, although EScaAG2 expression is lower than EScaAG1 expression.
Functional studies employing virus-induced gene silencing (VIGS) demonstrate that knock down of EScaAG1 and 2
function leads to homeotic conversion of stamens into petaloid structures and defects in floral meristem
termination. However, carpels are transformed into petaloid organs rather than sepaloid structures. We also show
that a reduction of EScaAG1 and EScaAG2 expression leads to significantly increased expression of a subset of floral
homeotic B genes.
Conclusions: This work presents expression and functional analysis of the two basal eudicot AG homologs. The
reduction of EScaAG1 and 2 functions results in the change of stamen to petal identity and a transformation of the
central whorl organ identity from carpel into petal identity. Petal identity requires the presence of the floral
homeotic B function and our results show that the expression of a subset of B function genes extends into the
central whorl when the C function is reduced. We propose a model for the evolution of B function regulation by C
function suggesting that the mode of B function gene regulation found in Eschscholzia is ancestral and the Cindependent regulation as found in Arabidopsis is evolutionarily derived.
Background
Flowers are complex structures composed of vegetative
and reproductive organs that are arranged in concentric
whorls in most angiosperms. The vegetative floral
organs, the sepals and the petals, develop in the outer
whorls while the inner whorls are composed of the pollen-bearing stamens and in the center carpels enclose
the ovules. The carpels are the last organs formed in the
flower and the floral meristem is consumed in the process of carpel development [1]. As described by the
* Correspondence: annette.becker@uni-bremen.de
1
University of Bremen, Fachbereich 02 Biology/Chemistry, Evolutionary
Developmental Genetics Group Leobener Str., UFT, 28359 Bremen, Germany
Full list of author information is available at the end of the article
ABCDE model, floral homeotic transcription factors act
in a combinatorial fashion to determine the organ identity primordia for the four distinct whorls: A + E class
genes specify sepal identity; A + B + E class genes act
together to determine petal identity; B + C + E class
genes specify stamen identity; C + E class genes together
define carpel identity, and C + D + E class genes specify
ovule identity [2,3]. Most of these homeotic functions
are performed by members of the MADS-box gene transcription factor family. AGAMOUS (AG), a C class gene
in Arabidopsis is necessary for specification and development of stamen and carpals, and floral meristem
determinacy [4]. The flowers of the strong ag-1 mutant
shows complete homeotic conversions of stamens into
© 2010 Yellina et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Yellina et al. EvoDevo 2010, 1:13
http://www.evodevojournal.com/content/1/1/13
petals and carpels into sepals and a recurrence of these
perianth organs in a irregular phyllotaxy [5].
Members of the AG subfamily of MADS box genes
have been identified in all major clades of seed plants
but not in more basal, seed-free lineages indicating
that the AG clade originated around 300 to 400 million
years ago in the common ancestor of gymnosperms
and angiosperms. In gymnosperm species, AG orthologs were found to be expressed in male and female
reproductive cones, which is reminiscent of the angiosperm expression in stamens and carpels [6-8]. Gene
family phylogenies reveal several duplication events
within AG clade of MADS box genes (Figure 1 [9,10]).
The first duplication event at the base of the angiosperm lineage led to the origins of the SEEDSTICK and
AG clades including ovule specific D class genes and
the carpel and stamen specifying C class genes, respectively [10]. A more recent duplication in the C-lineage
gave rise to the PLENA clade and euAG clade, the
FAR (Antirrhinum)
PMADS3 (Petunia)
AG (Arabidopsis)
SHP2 (Arabidopsis)
SHP1 (Arabidopsis)
PLE (Antirrhinum)
FBP6 (Petunia)
EScaAG1 (Eschscholzia)
EScaAG2 (Eschscholzia)
AqAG1 (Aquilegia)
ThdAG1 (Thalictrum)
AqAG2 (Aquilegia)
ThdAG2 (Thalictrum)
ZMM2 (Zea)
ZMM23 (Zea)
OsMADS3 (Oryza)
OsMADS58 (Oryza)
ZAG1 (Zea)
ZAG2 (Zea)
ZMM1 (Zea)
OsMADS13 (Zea)
OsMADS21 (Oryza)
ZMM25 (Zea)
EScaAGL11 (Eschscholzia)
FBP7 (Petunia)
FBP11 (Petunia)
STK (Arabidopsis)
GGM3 (Gnetum)
Figure 1 Simplified phylogeny indicating duplication events of
the AG lineage in angiosperms based on Zahn et al., 2006 [18].
Red branches denote euAG lineage genes, purple branches the PLE
lineage genes, yellow branches symbolize the basal eudicot lineage,
green branches denote the monocot C class genes and blue
branches denote D class genes. GGM3 represents the gymnosperm
lineage of AG homologs. The California poppy genes are marked in
red letters. The blue star symbolizes the C/D duplication event and
the purple star indicates the EuAG/PLE duplication.
Page 2 of 13
former containing the Arabidopsis SHATTERPROOF1
and 2 genes (SHP1 and 2), the latter AG. This duplication occurred after the ranunculids (basal eudicots in
the order Ranunculales) diverged from the lineage
leading to the core eudicots [9,11].
The Arabidopsis members of the PLENA clade, SHP1
and 2 are required for dehiscence zone differentiation in
the fruit and consequently for pod shattering [12,13].
Interestingly, PLENA itself, a gene in Antirrhinum
majus, is functionally more similar to AG than SHP1
and 2, and FARINELLI (FAR), the Antirrhinum AG
ortholog is required for pollen development. Both FAR
and PLENA are necessary for floral meristem determinacy in Antirrhinum [14,15].
Gene duplications and subfunctionalization have also
occurred in C-lineage of monocots, but independently
of the eudicot duplications (Figure 1). ZAG1 from maize
is required for floral meristem determinacy and ZMM2
is involved in stamen and carpel identity [16]. The rice
homologs OSMADS3 and OSMADS58 share common
functions, but also show a degree of subfunctionalization. While OSMADS3 plays a major role in stamen and
a minor role in carpel identity, OSMADS58 has a strong
influence on carpel identity and floral meristem determination [17]. Independent duplications of AG homologs have been inferred for other flowering plant
lineages, but functional analyses of duplicated AG
homologs are sorely lacking outside of model core eudicot and grass species.
Here we report functional data of the AG homologs of
the basal eudicot Eschscholzia californica (California
poppy, Papaveraceae) that belongs to Ranunculales, a
basal eudicot order. Basal eudicots are a sister grade leading to the more diverse core eudicot clade. Investigation
of species in this grade can shed light on the divergence
of monocots and eudicots and events that may have promoted diversification within the core eudicots.
Two AG homologs, EScaAG1 and EScaAG2, and a D
lineage homolog, EScaAGL11 , have been identified in
E. californica. EScaAG1 and EScaAG2 show similar
expression patterns, but EScaAG1 is expressed at a
much higher level than EScaAG2 [18]. The expression
patterns of both genes resembles that of AGAMOUS
(AG) in Arabidopsis except that the Eschscholzia poppy
AG orthologs are expressed earlier in the floral meristem [18,19].
This work presents an experimental investigation of
the EScaAG1 and EScaAG2 gene function employing
VIGS to manipulate transcript concentrations. We map
the expression of both genes in more detail than previously published and demonstrate that the down regulation of C function genes in E. californica leads to an
induction of some floral homeotic B genes in the fourth
floral whorl.
Yellina et al. EvoDevo 2010, 1:13
http://www.evodevojournal.com/content/1/1/13
Results
EScaAG1 and EScaAG2 are very similar in sequence and
expressed differentially
The two AG homologues of E. californica, EScaAG1 and
EScaAG2, share 66.6% and 61.1% amino acid sequence
identity to AG of Arabidopsis, respectively. These paralogs
are very similar throughout the open reading frame and in
the 5’untranslated region (UTR) with 75% identity at the
nucleotide level and about 81.7% at the amino acid level
(Additional file 1). When the two paralogues are compared
along their UTR and open reading frame, the EScaAG2
nucleotide sequence shows a 45 bp insertion and 14 bp
deletion in the 5’ UTR and a 10 bp deletion in the 3’ part
of coding region of EScaAG1 (data not shown).
Quantitative Reverse Transcriptase (RT)-PCR was carried out on cDNA derived from floral organs at anthesis,
young fruits, leaves, and buds of different developmental
stages to learn more about the differential expression of
EScaAG1 and EScaAG2 (Figure 2A). Both genes are
expressed in the reproductive organs of the flower, in
young fruits and in all tested stages of flower development. EScaAG1 and EScaAG2 are expressed in sepals,
petals, and leaves at extremely low levels. EScaAG1 is
highly expressed in stamens, carpels, young fruits and
later stages of flower development. EScaAG2 is generally
expressed at a lower level than EScaAG1 with the exception of stamen, where its expression is about 1.5 ×
higher than that of EScaAG1. In young fruits and during
bud development, EScaAG2 transcript abundance is very
low in comparison to EScaAG1.
The spatial expression patterns of EScaAG1 and 2
were additionally analyzed through in situ hybridizations
to obtain a more detailed picture of the expression
domains. However, as the open reading frames and
UTR’s of EScAG1 and EScaAG2 are highly similar, we
were unable to generate probes that could discriminate
between both genes. As a consequence, in situ hybridization patterns were nearly identical for these genes.
The only difference between the in situ hybridization
patterns was a much lower level of expression for
EScaAG2 (data not shown). In the following section, we
refer to the composite expression of EScaAG1 and
EScaAG2 as EScaAG1/2 expression patterns.
EScaAG1/2 gene expression was first observed in the
stage 2 bud before the gynoecium initiates and was visible as lateral domains in a few cells in the floral meristem where later the stamen primordia are initiated
(Figure 2B). In a stage 4 bud, the expression expands
uniformly in the floral meristem but is excluded from
the central primordium where later the gynoecium
arises (Figure 2C). By late stage 4, EScaAG1/2 expression becomes restricted to the boundaries between the
stamen anlagen with weak expression at the tip in the
Page 3 of 13
floral meristem just before gynoecium initiates (Figure
2D). In stage 6, strong expression is found in the region
adjacent to the placenta, the apical part of the medial
carpel wall and in the stamens (Figure 2E). Later in late
stage 6, EScaAG1/2 expression is restricted to the adaxial side of the gynoecium and in the stamens (Figure
2F). In transverse sections of the developing flower bud,
EScaAG expression is confined to the apical part of the
ovules but not in the placenta. In later stages of ovule
development, the EScaAG1/2 expression is stronger on
the adaxial than on the abaxial side (Figure 2G, H, I). In
summary, EScaAG1/2 genes are expressed during floral
meristem initiation at stage 2, during early development
of stamen and carpel primordia and later in the developing stamens and ovules.
EScaAG1 and EScaAG2 confer stamen identity
Virus induced gene silencing (VIGS ) was employed to
investigate the functions of EScaAG1 and EScaAG2 during flower development. This method allows transient
down-regulation of gene expression via modified plant
viruses, in our case the Tobacco Rattle Virus (TRV).
The E. californica flower is composed of a single sepal
occupying the first floral whorl, two whorls of four
petals and a varying number of stamen whorls ranging
from four to eight. The inner floral whorl produces a
bicarpellate gynoecium (Figure 3A) [20]. Overall, the
phenotypic effects of the EScaAG1 and EScaAG2 VIGS
were restricted to flowers. Treatment plants exhibited a
loss of stamen identity, homeotic conversion of stamens
into petals, and a loss of carpel characteristics. Additionally, EScaAG1 and 2 VIGS results in a loss of floral meristem termination. None of the analyzed pTRV2-E
(mock treatment, treated with the empty pTRV2 vector)
treated or untreated plants showed homeotic conversions or signs of loss of floral meristem termination, and
the vegetative habit also did not show any deviations
from untreated plants (Table 1 and [21]).
In total, 120 plants were infected with pTRV2EScaAG1, 120 plants with pTRV2-EScaAG2, another
120 plants were inoculated with pTRV2-EScaAG1/2,
and 12 plants were infected with pTRV2-E as a mock
control. The first three flowers of each plant were analyzed because the frequency of phenotype decreases in
the later formed flowers [21]. The phenotype scores for
each treatment are summarized in Table 1: 239 flowers
of plants infected with pTRV2-EScaAG1 were analyzed,
of which 122 flowers (51.0%) showed homeotic conversion in the third and fourth whorl floral organs. Of
these 122 flowers, 4.5% showed homeotic conversion of
all stamens into petal-like organs (Figure 3B). A total of
209 flowers of plants infected with pTRV2-EScaAG2
were observed, and of these 118 flowers (56.4%) showed
Yellina et al. EvoDevo 2010, 1:13
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relative expression
A
Page 4 of 13
EScaAG1
EScaAG2
2
B
C
fm
fm
se
sp
se
1
fm
0
ls
pa
se
t
pe
s
al
st
am
s
en
s
el
rp
a
c
es
av
le
g
un
yo
ts
ui
fr
1m
0-
m
ds
bu
2m
1-
m
ds
bu
G
F
3m
2-
m
ds
bu
m
m
>3
ds
bu
pl
ov
st
g
ov
p
pl
sp
st
se
pp
g
I
H
pl
E
D
p
se
gw
pl
ov
gw
se
Figure 2 Expression analysis of EScaAG1 and EScaAG2 in flowers of untreated plants shown by quantitative RT-PCR and in situ
hybridization. (A) Q-PCR based relative expression analysis of EScaAG1 and EScaAG2 in E. californica. Actin and GAPDH were used as reference
genes. (B) to (I) in situ hybridization pattern of the EScaAG genes using an EScaAG1 probe. (B) Longitudinal section of a bud in stage 2. (C)
Longitudinal sections of a bud in stage 3. (D) Longitudinal sections of a bud in stage 4. (E) Longitudinal section of a bud in late stage 6. (F)
Longitudinal section of a bud in stage 7. (G) Transverse section of a bud in stage 7. (H) Transverse section of a bud in stage 8. (I) Transverse
section of a bud in stage 9. All scale bars = 100 μm, abbreviations: fm, floral meristem; g, gynoecium; gw, gynoecium wall; ov, ovule; p, petal; pl,
placenta; pp, petal primordium; se, sepal; sp, stamen primordium; st, stamen.
homeotic transformation of stamens and carpels. Of all
flowers developing a silencing related phenotype, 15%
exhibited complete homeotic transformation of all stamens into petal-like organs (Figure 3C). Of the 261
flowers of plants infected with pTRV2-EScaAG1/AG2,
174 flowers (66.6%) showed homeotic transformation of
stamens and carpels and 15% of the latter exhibited
complete homeotic transformation of all stamens into
petal-like organs (Figure 3D-H, Table 1).
Interestingly, EScaAG1 and 2 VIGS-treated plants
exhibited conversion of stamen to petaloid organs in different stamen whorls (Table 1). Focusing on plants
infected with pTRV2-EScaAG1, 64 flowers (95.5% of the
flowers with homeotic conversion in the third whorl)
showed partial homeotic transformation of only the
outer whorls of stamens into petaloid organs (Figure 3I),
while the inner stamen whorls maintained a wild type
appearance. In contrast, 45 flowers (84.9% of the flowers
with homeotic conversions in the third whorl), from
plants infected with pTRV2-EScaAG2 showed homeotic
conversion of only the inner stamen whorls to petaloid
organs (Figure 3J). Plants infected with pTRV2EScaAG1/2 exhibited composite phenotypes: 96 flowers
(84.9% of the flower with homeotic conversion in the
third whorl) exhibited partial homeotic conversion of
outermost and innermost whorls while retaining wild
type stamen morphology in the central stamen whorls
(Figure 3K). Homeotic transformations of stamens into
petaloid organs occurred in various degrees as we
observed phenotypes ranging from complete petal-like
organs (Figure 3F) to mosaic staminoid-petaloid structures (Figure 3K).
Histological transverse sections of EScaAG1 and 2
VIGS-treated plants reveal further details of the homeotic conversions of stamens and gynoecia (Figure 3L, M).
In comparison to pTRV2-E treated plants (Figure 3M),
the connective of the stamens in the silenced plants is
elongated when compared to untreated plants and the
theca contain three pollen sacs in a few cases rather
than two as seen in untreated plants. The number of
vascular bundles in the connective is also increased
from one in untreated to five in stamens of VIGS treated plants. Additionally, the gynoecium in the center of
the flower of VIGS-treated plants is composed of two
fused parts reminiscent of petals. A solid ovary wall is
missing in the VIGS-treated plants as well as lateral differentiation of the ovary wall, such as a placenta or
ovules (Figure 3L, M).
Yellina et al. EvoDevo 2010, 1:13
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Page 5 of 13
C
B
A
D
G
F
E
st
ow
p
s
s
H
G
Fp
g
g
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J
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K
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ow
L
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s
p
ow
ow
p
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mw
iw
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iw
2
EScaAG1
pl
cw
st
1,5
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1
0,5
s
pst
st
p
0
cw
pT
U
RV
2:
AG
1pT
1
RV
2:
AG
1pT
2
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2:
AG
1pT
3
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2:
AG
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1
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2:
AG
pT
2RV
2
2AG
1/
pT
21
RV
2A
G1
/2
-2
M
relative expression
ov
Figure 3 Phenotypes of plants treated with pTRV1 and pTRV2-EScaAG1, pTRV2-EScaAG2, or pTRV2-EScaAG1/2 and expression analysis
of the VIGS treated plants. (A) Wild type phenotype of an E. californica flower treated with pTRV2-E. (B) Phenotype of an EScaAG1 VIGS treated
plant showing full homeotic conversions of stamens into petals. (C) Phenotype of an EScaAG2 VIGS treated plant showing full homeotic
conversions of stamens and carpels into petal-like structures. (D) Phenotype of a flower silenced for EScaAG1/2 showing homeotic conversions of
stamens into petals. (E) A mock treated plant with disassembled floral organs. (F) Disassembled flower of a plant treated with pTRV2-EScaAG1/2
showing homeotic conversions of stamens into petals. The same phenotype was also achieved with plants silenced for EScaAG1 or EScaAG2
individually. (G) Transverse hand section of a flower from a mock treated plant. (H) Transverse hand section of a flower from a plant silenced for
EScaAG1/2 showing homeotic conversions of stamens into petals. (I) Disassembled flower of a plant silenced for EScaAG1 showing partial
homeotic conversions of only the outer whorl stamens. (J) Disassembled flower of a plant treated with pTRV2-EScaAG2 showing that the inner
whorl of stamens is converted into petal-like structures. (K) Disassembled flower of a plant treated with pTRV2-EScaAG1/2 exhibiting homeotic
conversions of the innermost and outermost stamens whorls into petals while the middle whorls show mild deviation from wild type stamens
while the center whorl stamens remain more stamen-like. (L) Transverse section of a flower of an untreated plant. (M) Transverse section of a
flower from an EScaAG1 VIGS treated plant showing homeotic conversion of stamens into petals, petal-stamen mosaic structures, malformed
stamens and a gynoecium lacking tissue differentiation, ovules, and placenta. (N) Real-Time PCR analysis of the first bud of individual E.
californica plants treated with VIGS and untreated (U). Plants were treated with pTRV2-EScaAG1 are abbreviated as VIGS AG1, plants treated with
pTRV2-EScaAG2 as VIGS AG2. Numbers below indicated individual plants and the relative expression level of EScaAG1 and EScaAG2 in untreated
plants was set to 1. Abbreviations: cw, carpel wall; ov, ovule; p, petal; pl, placenta; se, sepal; pst, petaloid stamens; st, stamen.
Yellina et al. EvoDevo 2010, 1:13
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Page 6 of 13
Table 1 Overview of the observed phenotypes of EScaAG VIGS in California poppy
Phenotypes observed
pTRV1/
pTRV2-E
pTRV1/pTRV2EScaAG1
pTRV1/pTRV2EScaAG2
pTRV1/pTRV2EScaAG1+2
1
No. of inoculated plants
12
120
120
120
2
No. of analyzed flowers
36
239
209
261
3
No. of flowers showing phenotype in the third and fourth whorls
0
122 (51.0%)
118 (56.4%)
174(66.6%)
3.1
No. of flowers with homeotic conversions in the stamens
0
67 (54.9%)
53 (44.9%)
113 (64.9%)
3.1.1 No. of flowers with transformation of all the stamens into petals
0
3 (4.4%)
8 (15%)
17 (15%)
3.1.2 No. of flowers showing only outer stamen whorls converted into
petaloid organs
0
64 (95.5%)
0
0
3.1.3 No. of flowers showing only inner stamen whorls converted into
petaloid organs
0
0
45 (84.9%)
0
3.1.4 No. of flowers showing only outer and inner stamen whorls
converted into petaloid organs
0
0
0
96 (84.9%)
3.2
40 (22.9%)
0
27 (22.1%)
31 (26.2%)
3.2.1 No. of flowers with flattened green gynoecium
No. of flowers with alterations in the carpels
0
23 (85.1%)
26 (83.8%)
32 (80%)
3.2.2 No. of flowers with an orange pigmented gynoecium
0
4 (17.3%)
5 (16.1%)
8 (20%)
3.3
0
62 (50.8%)
80 (67.7%)
110 (63.2%)
No. of flowers showing defects in the floral meristem
termination
The strength of the observed phenotypes was correlated with the degree of reduction in EScaAG1 and 2
transcript levels as measured by Q-PCR. The first floral
bud (size 1 to 3 mm in diameter) of randomly selected
plants treated with the EScaAG1, EScaAG2, and
EScaAG1/2 VIGS vectors was collected and correlated
with the phenotype of the next formed flower. In 99%
of the cases (n = 414) we observed that when the secondarily formed flower showed a phenotype, the first
flower exhibited a phenotype as well. This consistent
pattern allowed us to predict the phenotype of the first
bud used for quantitative RT-PCR based on the second
flower’s phenotype (see also [21,22]). The changes in
EScaAG1 and EScaAG2 expression in the first buds
(1 to 3 mm bud diameter) of individual VIGS treated
plants are documented in Figure 3N. Targeted silencing
of individual EScaAG genes was not achieved, suggesting
that the overlap in observed phenotypes result from a
reduction of expression of both AG paralogs. Irrespective of the silencing vector used, EScaAG1 expression
was generally reduced from 70% to 10% of its wild type
expression and EScaAG2 expression was reduced from
25% to less than 5%. The use of the pTRV2-EScaAG1/2
vector resulted in similar reductions in expression levels
for both genes. While six plants show silencing of both,
EScaAG1 and EScaAG2, one plant (pTRV2:AG2-1) treated with EScaAG2-targeted VIGS exhibited reduction of
EScaAG2 expression but increased EScaAG1 expression
relative to untreated plants, demonstrating the variability
of VIGS experiments. However, we were able to show a
significant reduction of expression in six of seven randomly analyzed buds from individual plants.
VIGS of C-function genes results in homeotic conversions
of carpels into petal-like organs
In addition to homeotic conversions of stamens into
petal-like structures in plants infected with pTRV2EScaAG1 and pTRV2-EScaAG2, we observed changes to
the gynoecium morphology. The gynoecia of untreated
plants develop as round green cylinders and consist of
two fused carpels. This cylinder-like structure was disturbed in EScaAG1 and EScaAG2 VIGS-treated plants
and the gynoecia of the VIGS treated plants were transformed either into (i) flattened green structures lacking
ovules in some cases (Figure 4A Table 1) or (ii) flattened organs showing petal characteristics such as
orange pigmentation and petal-like epidermal surface
structure (Figure 4B Table 1). The latter was empty
(Figure 4A, B) or contained additional floral organs (Figure 4F Table 1).
In order to determine whether the petal-like pigmentation of the gynoecium was associated with a change in
cell surface morphology we conducted Scanning Electron Microscopy (SEM) analysis of the carpel whorl. In
the wild type, the carpel surface is composed of small
compact cells interrupted by stomatal cells (Figure 4C)
and the petal surface is composed of long and narrow
cells arranged in a parallel manner (Figure 3D) [20].
SEM micrographs of an orange-pigmented gynoecium
reveal a mosaic pattern of tubular petal-like cells next to
small compact cells typical for a carpel surface scattered
with stomata (Figure 3E). This indicates that the gynoecia of EScaAG1 and EScaAG2 VIGS treated plants not
only show a partially petal-like pigmentation but have
also acquired petal-like cell surface characteristics,
Yellina et al. EvoDevo 2010, 1:13
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supporting the hypothesis that these gynoecia are partially transformed into petal-like organs.
Treating poppy plants with EScaAG1 and 2 VIGS not
only resulted in the loss of stamen and carpel characteristics but also in the addition of petal organ identity to
the carpel whorl. We tested the hypothesisis that the
expression domains of floral homeotic B genes was
extended to the central gynoecium whorl in EScaAG1
and 2 VIGS treated plants using real-time PCR to assess
expression of the three poppy floral homeotic B class
genes EScaDEF1, EScaDEF2, and EScaGLO at anthesis
and pre-anthesis (Figure 4G, H). B and C gene expression in untreated gynoecia was also characterized
(Figure 4D). As expected EScaAG1 as well as EScaAG2
were expressed in gynoecia before and at anthesis. Surprisingly, the class B gene ortholog, EScaDEF1 was
expressed in gynoecia at a comparatively high level,
although expression levels of two other B-class genes
EScaDEF2 and EScaGLO were hardly detectable. Next,
the expression of class B and C genes was recorded in
the gynoecia of VIGS treated plants (Figure 4G). The
relative expression of all analyzed genes was normalized
by setting levels to one in gynoecia of untreated plants
before anthesis. In the gynoecia of VIGS treated plants
(Figure 4H), expression of EScaAG1 was reduced to 50%
and even 20% in the gynoecia of VIGS treated plants
and expression of EScaAG2 was reduced in most gynoecia as well. VIGS treatments had no impact on EScaDEF1 expression in the gynoecia. However, the
expression of EScaDEF2 was drastically increased
between 5.8-fold and 17.7-fold relative to expression in
untreated gynoecia. Transcript abundance of EScaGLO,
also increased significantly upon silencing of C function
genes by 2.2 to 5.7 times in the EScaAG1 and EScaAG2
VIGS treated plants. These expression analyses indicate
that in central whorl organs with reduced expression of
C function genes, two B function genes EScaDEF2 and
EScaGLO were expressed at significantly higher level in
EScaAG1 and EScaAG2 VIGS treated than in untreated
or mock treated plants.
For the Arabidopsis B proteins APETALA3 (AP3) and
PISTILLATA (PI) it was shown that their homeotic
function requires the formation of AP3-PI heterodimers
[23]. EScaDEF2 is an AP3 homolog while EScaGLO is
the PI homolog [24] and simultaneous upregulation of
the AP3 and PI orthologs in poppy suggests that they
might form heterodimers in the central whorl of C function silenced flowers and cause the observed homeotic
gynoecium-petal conversions.
EScaAG1 and EScaAG2 are involved in the regulation of
floral meristem termination
The flowers of the plants treated with EScaAG1 and
EScaAG2 VIGS showed not only homeotic conversions
Page 7 of 13
of stamens into petaloid organs, petal-like features in
the central whorl, and a reduction in ovule number, but
also signs of prolonged floral meristem activity. All treated plants showed increases in floral organ number in
the stamen and central whorls. Moreover, flowers exhibiting a strong silencing phenotype showed ectopic
structure enclosed inside the gynoecium whorl ranging
from carpel like leaves to additional gynoecia and ectopic flowers (Figure 4F).
Interestingly, we observed a significant increase in stamen number in the weaker floral phenotypes characterized by no obvious homeotic organ conversions (Table 2).
Untreated plants produced 26.2 stamens per flower on
average, EScaAG1 VIGS-treated plants without any
homeotic conversions developed 29 stamens per flower,
EScaAG2 VIGS-treated produced 28.2, and plants treated
simultaneously with EScaAG1/2 produced 28.6 stamens
on average. This suggests that whereas a mild reduction in
EScaAG1 and EScaAG2 expression may not affect floral
organ identity any reduction in expression can induce an
increase in stamen number.
Discussion
This study is the first functional analysis of floral
homeotic C function genes in a basal eudicot. We
employed VIGS to transiently down-regulate EScaAG1
and EScaAG2 in E. californica and observed homeotic
conversions of stamens into petals, reduced floral meristem termination, and transformation of the gynoecium
into petal-like structures. EScaAG2 is expressed at lower
levels (also observed by [18]) but despite the reduced
expression of EScaAG2, molecular evolutionary analyses
failed to detect evidence of reduced evolutionary constraint (see below).
The two AG paralogs of E. californica, EScaAG1 and
EScaAG2 are quite similar on both protein and nucleotide level including the 5’UTR region indicating that
they are duplicates. Generally it is hypothesized, that
duplicated genes will not persist over evolutionary time
unless sub-, or neofunctionalization results in functional
divergence [25-27]. EScaAG1 and EScaAG2 share about
81.7% sequence similarity in the open reading frame and
are 75.5% identical when the 5’UTR is included. The
origin of these paralogs may be associated with an
ancient whole genome duplication event that has been
inferred on the lineage leading to Eschscholzia [28].
Using a penalized likelihood approach [29] we estimated
an age of 51 million years for the EScaAG duplication.
This divergence time was obtained using a maximum
likelihood tree for the AG subfamily [30] calibrated with
taxon ages reported in [31].
No evidence of reduced constraint on EScaAG2 was
inferred from analysis of the ratio of nonsynonymous to
synonymous nucleotide substitutions on the branch
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Figure 4 Carpel whorl phenotype of EScaAG silenced plants and expression analysis of floral homeotic genes. (A) Gynoecium of an
untreated plant (left) and of a plant silenced for EScaAG2 (right) (B) Flat orange gynoecium without ovules of a plant treated with pTRV2EScaAG1. (C) Scanning electron micrograph (SEM) of the wild type gynoecium surface structure. (D) SEM of a wild type petal surface structure.
(E) SEM of the central floral whorl organ of a plant treated with pTRV2-EScaAG1 showing a mix of petal and gynoecium surface structures. (F)
The central whorl floral organ (pg, for petaloid gynoecium) of a plant treated with pTRV2-EScaAG2 showing petaloid and carpeloid features as
well a lack of ovules. This organ encloses an ectopic flower consisting of a remnant gynoecium (g), petals (p) and stamens (st). (G) Relative
expression of class B and C genes in young carpels before anthesis and mature carpels at anthesis of untreated plants, (H) Real-Time RT-PCR
expression analysis of EScaAG1, EScaAG2, EScaDEF1, EScaDEF2, and EScaGLO in the gynoecia of VIGS treated plants. Abbreviations used in (G) and
(H): yc, young carpel before anthesis; mc, mature carpel at anthesis; u, untreated plants; pTRV2-E, plants treated with pTRV1 and pTRV2-E; pTRV2AG1, plants silenced for EScaAG1; pTRV2-AG2, plants silenced for EScaAG2; pTRV2-AG1/2, plants silenced for EScaAG1 and EScaAG2.
Table 2 Stamen numbers of in EScaAG1, EScaAG2, and EScaAG1/2 VIGS treated plants
Untreated/pTRV1 and pTRV2- EScaAG1 VIGS
E treated
treated
EScaAG2 VIGS
treated
EScaAG1/2 VIGS
treated
No. of flowers analyzed
28
244
242
333
No. of flowers without homeotic conversions
28
93
123
92
Average no. of stamens in flowers without
homeotic conversions
26.2 ± 1.9
29* ± 4.1
28.2* ± 2.9
28.6* ± 4.5
* Significant change to untreated control plants (ANOVA test)
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leading to EScaAG2 [30]. A recent shift in constraint
on EScaAG2 may not be detectable [32], but the molecular evolutionary analyses indicate that both EScaAG2
and EScaAG1 have been evolving under selective constraint for much of the approximately 50 million years
since duplication. These results suggest that both
EScaAG1 and 2 have been selectively maintained in the
lineage leading to E. californica.
Gymnosperm and angiosperm AG homologs are
highly conserved but gene duplications have spurred
functional diversification. The observation that knocking
down EScaAG1 and 2 individually results in overlapping
phenotypes can be explained by two alternative scenarios. First, the two poppy AG paralogs may be working
redundantly in the specification of floral organ identity
and floral meristem determinacy. Alternatively, the
VIGS method may not be able to individually silence
paralogs with highly similar sequences. Our results are
not fully consistent with either of these interpretations.
Expression analyses of single knock down VIGS plants
showed that transcript abundance of both genes was
decreased, but EscaAG2 was silenced more strongly
than EscaAG1. With respect to the first scenario, the
selective maintenance of fully redundant genes over 50
million years is highly unlikely. Full knockouts (vs.
knock downs) for each paralog may be required to
reveal subtle functional divergence.
Differences in expression between EScaAG1 and
EScaAG2 (Figure 2A) and deviations in spatial distribution of the homeotic conversions of stamens into petals
(Figure 3I-K) hint at some degree of subfunctionalization. However, we were not able to relate the distinct
phenotypes of only outer stamen whorl homeotic conversions in the case of EScaAG1 VIGS and only inner
stamen whorl conversion in the EScaAG2-silenced flowers to the expression EScaAG1 and EScaAG2 expression
data. In almost all analyzed floral buds we have simultaneous down-regulation of both genes with always a
higher residual EScaAG1 expression than EScaAG2
expression, suggesting that subtle spatial expression difference at a very early developmental stage might play a
role which we were not able to detect with our expression analysis. A less transient approach such as stable
transformation with hairpin RNA constructs that would
be able to silence EScaAG1 and EScaAG2 expression
individually is required to rigorously characterize functional domains for EScaAG1 and 2, and test the subfunctionalization hypothesis.
Another characteristic of the EScaAG1 and 2 VIGS
phenotype is the loss of carpel organ identity. The most
common phenotype observations were flattened green
gynoecia or flat petaloid gynoecia showing an orange
pigmentation and cell surface structure typical for petals
(Figure 4A-E, Additional file 2.). The latter finding
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indicates that in E. californica, homeotic conversions of
gynoecia into petaloid structures can occur when the C
function is missing. This homeotic conversion coincides
with the expansion of the expression domains of two
class B genes, EScaDEF2 and EScaGLO, into the central
floral whorl of EScaAG1 and EScaAG2 VIGS treated
plants. The third B class gene, EScaDEF1 is also
expressed in the gynoecia of untreated plants and
expression levels are unaffected by reduction of C class
gene expression in VIGS treated plants (Figure 5). These
findings suggest that EScaDEF1 expression is independent of class C gene expression while EScaDEF2 and
EScaGLO are negatively regulated by class C genes in
the central floral whorl.
Interestingly, EScaDEF2 and EScaGLO are expressed in
parallel with the class C genes in the stamen whorl
which indicates C independent expression of the two
class B genes in stamen whorls in contrast to C dependent expression in the central floral whorl. Thus, a
cofactor (X) restricted to the central whorl can be postulated to inhibit expression of EScaDEF2 and EScaGLO
expression along with the C class proteins EScaAG1 and
EScaAG2 (Figure 5).
This type of C-dependent regulation of B class genes
is in contrast to the strong Arabidopsis ag-3 mutant,
where full homeotic conversions of stamens into petals
and carpels into sepals are observed. Even in the weaker
ag-4 mutant, the carpel is not converted into a petal-like
structure, but rather into a sepal [33]. Single or double
mutants shp1/shp2 do not show any floral homeotic
functions in Arabidopsis. Phenotypic effects are detectable only after fertilization [34]. In contrast, the Antirrhinum ple-1/far double mutant shows the type of
floral homeotic conversions we observe in poppy: carpels are converted into petal-like structures and additional flower enclosed inside the fourth whorl unlike in
the third whorl in Arabidopsis [15]. In the Arabidopsis
ag mutant, the expression of the B function genes AP3
and PI in the fourth whorl is prevented by the action of
SUPERMAN (SUP) [35] and carpels are converted to
sepal-like organs [36]. Therefore, it seems that the regulation of B function genes is independent of C class
gene function in Arabidopsis. However, at this point we
cannot exclude the hypothesis that AG together with
the closely related SHP1 and SHP2 genes work with
SUP to repress B gene expression in the fourth whorl.
In the Antirrhinum ple-1/far double mutant, an expansion of the B function expression domain towards the
fourth whorl was observed as a result of a C function
reduction. It was suggested that the putative SUP orthologs in Antirrhinum, OCTANDRA (OCT) requires PLE
or FAR to exclude B function gene expression from the
fourth whorl while SUP in Arabidopsis acts independently of AG [15].
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EScaDEF2
EScaGLO
Page 10 of 13
+ X?
EScaDEF1
EScaAG1
EScaAG2
?
EScaAGL2
EScaAGL9
sepals
petals
stamens
carpels
Figure 5 Hypothesis on the regulation of class C dependent B
gene expression in E. californica. This modified BCE model of E.
californica floral organ identity specification includes the B class
genes (orange boxes) EScaDEF1, EScaDEF2, and EScaGLO that are
supposed to be expressed in second and third (stamen) whorl.
Expression of EScaDEF1 is also found in the central whorl. Two class
C genes (green boxes) are expressed in the stamen and central
whorl, and the two class E genes EScaAGL2 and EScaAGL9 (blue
boxes) are expressed in all whorls except for the sepal whorl for
EScaAGL9 [43] while no information is available on the expression
domain of EScaAGL2. Red bars indicate repression of gene
expression. It remains unclear by which mechanism EScaAG1 and
EScaAG2 expression is restricted to the reproductive whorls of the
flower and if repression of EScaDEF2 and EScaGLO by the two class
C genes is direct or mediated by a co-factor. An A function has not
yet been described in California poppy.
Our analysis of the EScaAG1 and EScaAG2 VIGS
flowers suggests that the regulation of poppy B function
genes is more similar to Antirrhinum than to Arabidopsis because we also find an expansion of petal-like tissues specified by the B function in the fourth whorl. As
postulated for Antirrhinum, the negative regulation of B
function genes in the fourth whorl may involve the activation of an E. californica SUP ortholog. A poppy SUP
ortholog could be positively regulated by EScaAG1 and
2 or interact with these genes to restrict B function
expression to the second and third whorl in wild type
plants.
The fact that B gene expression is restricted by C
function in E. californica as a representative of a basal
eudicot lineage and Antirrhinum, a member of the
asterid clade, in contrast to C independent regulation in
Arabidopsis indicates that the former regulatory scenario
might be ancestral. However, C function dependent regulation of B class genes has also not been reported in
monocots such as rice. Down regulation of the rice AG
homolog, OsMADS58, did not result in expansion of the
expression domains for B class genes and carpel to lodicule transformation have not been observed in osmads3
mutants or OsMADS58 RNAi lines [17].
This suggests three possibilities for the evolution of
class C dependent regulation of class B gene expression:
(i) This type of regulation had evolved before the monocot and eudicot lineages diverged but was lost independently, in lineages leading to Arabidopsis and rice. (ii)
The C-dependent regulation of B expression evolved
once in the eudicots before the divergence of Ranunculales and was lost in the lineage leading to Arabidopsis
after their split from the asterids. (iii) Class C genes
were recruited twice independently, once in the lineage
that led to E. californica after it diverged from the rest
of the dicots and a second time in the lineage leading to
Antirrhinum after its divergence from the lineage leading to Arabidopsis. Since class C floral homeotic
mutants are not yet available from basal angiosperms or
non-grass monocots all three of these scenarios are
equally parsimonious.
As reported for Arabidopsis ag mutants, a reduction of
EScaAG1 and 2 function in E. californica leads to
defects in floral meristem termination, albeit in a more
complex pattern than observed in Arabidopsis. The stamen whorls of EScaAG1 and 2 VIGS-treated plants are
more numerous than in the control plants even if the
phenotype is mild, for example, no homeotic conversions of reproductive organs (Figure 3F). These observations support inferences drawn from work on A.
thaliana and A. majus where mild reductions in C-function affect floral meristem determinacy [37,38]. The
morphogenesis of E. californica flowers differs from
most core eudicots, for example, Arabidopsis, in that the
innermost stamen whorls are still being formed when
the central gynoecium is initiated. A ring of cells with
meristematic activity around the gynoecium is maintained while the central floral meristem is consumed in
the process of gynoecium initiation [20]. This suggests
that a mild reduction in EScaAG1 and 2 expression is
sufficient for a prolonged meristem activity in this ring
shaped meristem that produces additional stamen
whorls in EScaAG1 and 2 VIGS-treated flowers. Additionally, our results suggest that EScaAG1 and 2 regulate
the termination of meristem activity in E. californica.
Regulation of meristematic activity was observed in the
central floral meristem and the ring meristem that gives
rise to stamen whorls independently of the ceasing central floral meristem activity (Table 2). The influence of
EScaAG1 and 2 VIGS on the stamen whorls is especially
interesting as stamen numbers in wild type E. californica
are phenotypically variable, ranging from 18 up to 34
stamens when individuals are grown under identical
conditions and constant light [20]. Even slight differences in the timing and dose of EScaAG1 and EScaAG2
transcript abundance between plants could account for
these stamen number variations in wild type plants. The
number of stamens in E. californica generally coincides
with the plant’s stature: as has been reported for Stellaria media (chickweed) [39], healthier plants produce
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more stamens. Our analyses suggest that the number of
stamens produced is dependent on the amount of
EScaAG1 and EScaAG2 transcript in E. californica flowers. This might indicate a stature-dependent regulation
of class C floral homeotic genes in the ring-like meristem. Moreover, a direct link could exist between floral
homeotic gene action and male fecundity in natural
populations.
This additional function of the class C genes in E.
californica in the zone of meristematic activity around
the gynoecium might represent a more general mode of
function for class C genes in the large subgroup of
angiosperms with several stamen whorls and often varying stamen numbers. The duration of class C genes
activity in the meristems generating these reiterating
stamen whorls might also determine stamen number in
these species.
Our study on the function EScaAG1 and EScaAG2 in
E. californica reveal that the VIGS method is suitable to
analyze the evolution of gene regulation by enabling
gene function analysis in non-model plants for which
transgenic approaches are difficult to achieve. This work
shows that gene function and the regulation of floral
homeotic genes vary among plant lineages. Looking forward, the importance of VIGS for assessing gene function in non-model species will increase as advances in
sequencing technologies result in full transcriptome and
even genome sequences for an expanding number of
species sampled across the plant tree of life. While
sequence data will allow characterization of amino acid
conservation and gene duplication events, functional
studies in non-model species will be required to elucidate the evolution of regulatory networks influencing
flowering time and floral form over angiosperm history.
Materials and methods
Expression analysis
Q-PCR
Q-PCR assays were performed on floral organs, leaves,
young fruits, and buds of different developmental stages
in wild type plants. For expression analysis of the
EScaAG1 and EScaAG2 VIGS-treated plants, a single
bud (1 to 2 mm in diameter) was examined. For the
analysis of class C and B genes in VIGS-treated plants,
single gynoecia of either buds of 5 to 8 mm diameter
(young carpel) or from open flowers (mature carpel)
were collected. All samples were analyzed in three technical replicates. One μg of total RNA was reverse transcribed into cDNA using random hexamer primers and
the SuperScript III Kit (Invitrogen, Karlsruhe, Germany).
A total of 5 μl of 1:50 diluted cDNA was used as a template. DEF1 and Actin primers were designed with the
help of the UPL probe program (Roche, Mannheim,
Germany); all other primers sets were designed with one
Page 11 of 13
intron spanning primer (primer details are in Additional
file 3). Paralog specific primer pairs consist of forward
primers spanning at least one intron and a reverse primer spanning the deletion part of EScaAG1 in 3’ coding
region were used to discriminate between EScaAG1 and
EScaAG2 and the PCR product was sequenced to confirm primer specificity and the primer melting curves
were analyzed. Eschscholzia Actin2 and GAPDH were
used as reference genes. The Real-Time PCR reaction
mix consisted of: 5 μl of cDNA (1:50 dilution), 10 μl of
SYBR Green mix (Roche) and 0.8 to 1.2 pM primers.
The UPL Real-Time PCR mix consisted of 5 μl of 1:50
diluted cDNA, 100 nM UPL probe (Roche, #132 for
EScaDEF1 and #136 for Actin) and 0.04 pM of each primer. Real-Time PCR was performed using a Light
Cycler 480 (Roche) with the following cycle conditions:
initial heating of 95°C for 5 minutes, and 45 cycles of
10 s at 95°C, 10 s at 60°C and 10 s at 72°C. Cp values
were analysed according to the Genorm manual and
accurate normalization was carried out by geometric
averaging of multiple internal control genes [40].
In situ hybridisation
Non-radioactive in situ hybridization followed essentially
the protocol of [41]. The EscaAG1 and EScaAG2 coding
regions were cloned into the pDrive vector (Qiagen,
Hilde, Germany), the digoxigenin-labelled RNA probes
were transcribed using SP6 polymerase (Roche) and subsequently hybridized to floral tissue sections.
Virus-induced gene silencing
A 395 bp fragment of EScaAG1 was amplified from the
EScaAG1 coding region by using the primers VIGSEcAG1A to add a BamHI restriction site to the 5’ end of
the PCR product and EcAG1VIGS to add an XhoI
restriction site to the 3’ end (primer sequences reported
in Additional file 3). The amplicon was digested with
BamHI and XhoI and cloned into a similarly cut pTRV2
vector [42]. A 477 bp fragment of EScaAG2 was amplified from the EScaAG2 coding region by using the primers VIGSEcAG2A to add a BamHI restriction site to
the 5’ end of the PCR product and EcAG2VIGS to add
an XhoI restriction site to the 3’ end. The amplicon was
digested with BamHI and XhoI and cloned into a similarly cut pTRV2. pTRV2-EScaAG1/AG2 was constructed
by a 190 bp fragment of EScaAG1 was amplified from
the EScaAG1 coding region by using the primers XbaVIGSEcAG1Bfw to add a XbaI restriction site to the 5’
end of the PCR product and EcAG1VIGSXhorev to add
a XhoI restriction site to the 3’ end. The amplicon was
digested with XbaI and XhoI. A 214 bp fragment of
EScaAG2 was amplified from the EScaAG2 coding
region by using the primers EcoVIGSEcAG2Afw to add
an EcoRI restriction site to the 5’ end of the PCR product and EcAG2VIGSXbarev to add an XbaI restriction
site to the 3’ end. The amplicon was digested with
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EcoRI and XbaI and was then ligated together with the
EScaAG1 fragment into the EcoRI and XhoI cut pTRV2
vector producing the pTRV2-EScaAG1/AG2 plasmid.
The vector inserts of the double construct were confirmed by restriction digestion and sequencing. The
resulting plasmids were sequenced and transformed into
Agrobacterium tumefaciens strain GV3101. The agroinoculation was performed by injecting the Agrobacterium suspension into the shoot apical meristem as
described by [22].
Scanning electron microscopy and histology
Gynoecia of EScaAG1 and EScaAG2 VIGS-treated and
untreated plants were analyzed by Scanning Electron
Microscopy [14] for changes in the cell surface structure.
The gynoecia were incubated in 100% methanol for 10
minutes and subsequently for 10 minutes in 100% ethanol. Then they were kept overnight at room temperature
in 100% ethanol and dried with a Critical Point Dryer,
gold coated, and examined under the SEM (ISI-100B,
International Scientific Instruments, Pleasanton, CA,
USA). First formed buds of 1.6 to 2.5 mm in diameter
were collected for histological analysis and stained with
Safranin and Fast Green as described by [22].
Additional material
Additional file 1: Supplemental Figure 1: Alignment of the EScaAG1
and EScaAG2 protein sequences. Amino acids identical between two
paralogs are indicated by dots; dashes indicate deletion of five amino
acids located in the C-terminal region of EScaAG2. Dissimilar residues are
indicated by the respective amino acids.
Additional file 2: Supplemental Figure 2: Phenotypes observed in
the gynoecium of EScaAG VIGS treated plants. The Y-axis denotes the
percentages of different carpel identity phenotypes obtained by VIGS
(pTRV2-EScaAG1, n = 239; EScaAG2, n = 209, EScaAG1/2, n = 261 flowers).
Differently treated VIGS plants are shown on the X-axis. The green color
indicates the occurrence of flat green gynoecia; the orange color
symbolizes flat orange gynoecia. Stripes indicate gynoecia enclosing
ovules, plane color indicates a gynoecium lacking ovules, and the dotted
pattern indicates additional organs enclosed by the gynoecium.
Additional file 3: Supplemental Table 1: Sequences of primers used
in this study.
Abbreviations
AG: the floral homeotic C function gene AGAMOUS of A. thaliana; AP3: the
floral homeotic B function gene APETALA3 of A. thaliana; EScaAG1: E.
californica ortholog of AG; EScaAG2: E. californica ortholog of AG; EScaAGL
11: E. californica ortholog of the the ovule specific gene SEEDSTICK (formerly
known as AGL11) of A. thaliana; EScaDEF1: E. californica ortholog of the A.
majus floral homeotic B function gene DEFICIENS; EScaDEF2: E. californica
ortholog of the A. majus floral homeotic B function gene DEFICIENS ;
EScaGLO: E. californica ortholog of the A. majus floral homeotic B function
gene GLOBOSA; FAR: floral homeotic C function gene FARINELLI in A. majus;
OCT: putative stamen and carpel boundary specifying gene OCTANDRA in A.
majus; OSMADS3: floral homeotic C function gene of O. sativa; OSMADS58:
floral homeotic C function gene of O. sativa; PI: the floral homeotic B
function gene PISTILLATA of A. thaliana; PLE: the A. majus floral homeotic C
function gene PLENA; Q RT-PCR: Quantitative Reverse Transcriptase
polymerase chain reaction; SEM: Scanning electron microscopy; SHP: the
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SHATTERPROOF gene of A. thaliana; SUP: the stamen and carpel boundary
specifying gene SUPERMAN of A. thaliana; TRV: Tobacco rattle virus; UPL:
Universal probe library; UTR: Untranslated region; VIGS: Virus induced gene
silencing; ZAG1: floral homeotic C function gene of Z. mays; ZMM2: floral
homeotic C function gene Z. mays.
Acknowledgements
We thank Zsuzsanna Schwarz-Sommer as well as David Smyth for valuable
discussions about the VIGS phenotypes and S.P. Dinesh-Kumar for providing
the pTRV vectors. We thank the Becker lab members for lively discussions,
Teja Shidore and Gitanjali Darmadhikari for their help with the phenotyping.
We also thank Werner Vogel and Angelika Trambacz for poppy plant growth
and care. This work was made possible with funding from the German
Research Foundation (DFG) grant to A. B. (BE 2547/6-1, 6-2) and funding by
the University of Bremen. This paper is dedicated to the memory of the late
Zsuzsanna Schwarz-Sommer whose interest in this project was warmly
appreciated, and whose contribution to the field was inspirational.
Author details
1
University of Bremen, Fachbereich 02 Biology/Chemistry, Evolutionary
Developmental Genetics Group Leobener Str., UFT, 28359 Bremen, Germany.
2
Department of Plant Biology, University of Georgia, Athens, GA 30602-7271,
USA.
Authors’ contributions
AYL participated in the expression analysis and phenotype characterization.
SL carried out the cloning work and participated in expression analysis and
phenotype characterization. SO carried out and interpreted the in situ
hybridizations. RE analyzed and interpreted the Real-Time PCR data. JLM
performed sequence divergence time estimates and molecular evolutionary
analyses and edited the manuscript. AB conceived of the study, participated
in its design and coordination. AYL and AB wrote this manuscript. All
authors read and commented on drafts of the manuscript and approved the
final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 7 May 2010 Accepted: 1 December 2010
Published: 1 December 2010
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doi:10.1186/2041-9139-1-13
Cite this article as: Yellina et al.: Floral homeotic C function genes
repress specific B function genes in the carpel whorl of the basal
eudicot California poppy (Eschscholzia californica). EvoDevo 2010 1:13.
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